Attenuated ibv with extended cell culture and tissue tropism

ABSTRACT

The present invention relates i.a. to an IBV (infectious bronchitis virus) deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof and any IBV having all of the identifying characteristics of the deposited IBV. Further, the present invention relates to an immunogenic composition comprising said deposited IBV.

BACKGROUND OF THE INVENTION

Avian coronavirus infectious bronchitis virus (IBV) is the prototypegammacoronavirus of the family Coronaviridae, order Nidovirales.Infectious bronchitis virus infects the upper respiratory epithelium ofchickens, causing a respiratory disease, commonly complicated bysecondary bacterial pathogens (Cook et al. 2012. Avian Pathol.41:239-250). Some IBV strains additionally affect the renal tubuli,oviduct and parts of the gastrointestinal tract, leading to pathologicallesions and clinical symptoms in these organ systems. The virus has aworldwide presence in both commercial and backyard chicken. Due to itshigh genomic variability IBV is discriminated in a wide variety ofgeno-, sero- and protectotypes. IBV is currently regarded as one of theeconomically most relevant viral pathogens in the poultry industry.

Infectious bronchitis virus is an enveloped virus with a positive sensesingle-stranded RNA genome of 27.6 kb (Cavanagh 2007. Vet. Res.38:281-297). The first two-thirds of the viral genome comprise a largecoding region (also designated as gene 1), divided into two open readingframes 1a and 1b, which encode for at least 15 nonstructural proteinsinvolved in RNA replication, editing, and transcription. The lastone-third of the viral genome codes for structural proteins: the spikeprotein (S, encoded by gene 2), the envelope protein (E, encoded by gene3c), the membrane protein (M, encoded by gene 4), and the nucleocapsidprotein (N, encoded by gene 6). Proteins S, E and M are part of theviral envelope while protein N forms the ribonucleoprotein core alongwith the viral RNA. The coronavirus spike protein determines the hostspecies tropism (Kuo et al. 2000. J. Virol. 74:1393-1406). It is adimeric or trimeric transmembrane protein, which is proteolyticallycleaved into two subunits, S1 and S2. The glycosylated 51 domain formsthe ‘head’ of the spike protein and contains the receptor binding domainthat interacts with 2,3-linked sialic acids on the host cell surface(Promkuntod et al. 2014. Virology. 448:26-32). The S2 domain is theremaining part of the ectodomain (the ‘stalk’), the transmembrane domainand an endodomain located in the cytoplasm.

The to date widely used live-attenuated IBV vaccine strains H52 and H120were developed in the 1960s in the Netherlands, by serial passaging of aMassachusetts serotype IBV strain in embryonated chicken eggs (Bijlengaet al. 2004; Avian Pathol. 33:550-557). Said vaccine strains also haveto be propagated in embroynated chicken eggs for vaccine production.Today, IBV vaccines (both, inactivated and live vaccines) are stillpropagated in embryonated chicken eggs which is cumbersome andexpensive.

The only cell-line adapted IBV described so far is the IBV strainBeaudette, which efficiently replicates in Vero and BHK cells. Casais etal. 2003 (J. Virol. 77; 9084-9089) show that the S protein of Beaudetteis the determinant of cell line tropism by generating recombinant IBVsusing ectodomain sequences of the Beaudette spike, which were able totransfer this extended cell line tropism to another IBV (M41). Fang etal. 2005 (Biochemical and Biophysical Research Communication 336; pages417 to 423) disclose that the adaption of Beaudette for propagation inVero cells resulted in 49 amino acid modifications, 26 located withinthe spike protein. However, recombinant IBVs with Beaudette spike arenot suitable as vaccines. Ellis et al. 2018 (J. Virol. 92(23)) describethat recombinant Beaudette constructs with chimeric spikes withheterologous 51 subunits from M41 or QX in combination with Beaudettespike S2 subunit do not confer sufficient protection against 51homologous challenges. Also, Beaudette wild type does not provideprocection against homologous challenge like other licensed vaccinesbelonging to the Massachusetts serotype (Hodgson et al 2004: J Virol78:13804-13811 or Geilhausen et al 1973: Archiv für die gesamteVirusforschung 40: 285-290).

Taken together, providing IBV vaccines having an extended cell or tissuetropism by exchanging the spike protein to a heterologous Beaudettespike protein (recombinant IBV) would not result in IBV vaccinesproviding sufficient efficacy, and with the Beaudette spike sequencewould be limited to protection against a Massachusetts serotype strainchallenge and missing cross protection against further genotypes.Further, the IBV of the present invention is not a GMO (geneticallymodified organism) in contrast to the recombinant IBVs disclosed in theprior art.

Consequently, there is a need for efficient IBV vaccines having anextended cell or tissue tropism for efficient production. Preferably,such new IBV vaccines with extended cell or tissue tropism arenon-recombinant vaccines.

DETAILED DESCRIPTION OF THE INVENTION

Before the aspects of the present invention are described, it must benoted that as used herein and in the appended claims, the singular forms“a”, “an”, and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “an antigen”includes a plurality of antigens, reference to the “virus” is areference to one or more viruses and equivalents thereof known to thoseskilled in the art, and so forth. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, the preferred methods, devices, andmaterials are now described. All publications mentioned herein areincorporated herein by reference for the purpose of describing anddisclosing the cell lines, vectors, and methodologies as reported in thepublications which might be used in connection with the invention.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate such disclosure by virtue of prior invention.

Composition of Matter

The present invention solves the problems inherent in the prior art andprovides a distinct advance in the state of the art.

Generally, the present invention provides an IBV (infectious bronchitisvirus) deposited with the BVR of IZSLER under accession number DPS RERSCIC 16, any descendant IBV thereof or any IBV having all of theidentifying characteristics of the IBV deposited under DPS RE RSCIC 16.

Further, the present invention provides an IBV (infectious bronchitisvirus) deposited with the BVR of IZSLER under accession number DPS RERSCIC 16, any attenuated descendant IBV thereof having an extended cellor tissue tropism and being protective against virulent M41 challenge orany attenuated IBV having an extended cell or tissue tropism and beingprotective against virulent M41 challenge.

Advantageously, the experimental data show that the deposited strain andits decendants have an extended cell or tissue tropism, they are capableto infect and/or replicate in different cell lines and tissue cells.Surprisingly, the deposited strain and its decendants maintain vaccineefficacy until at least several passages, but have an improved safety.

The deposited IBV (IB66HP) strain is deposited at the BVR of IZSLER(Biobank of Veterinary Resources of the Istituto ZooprofilatticoSperimentale della Lombardia e dell'Emilia Romagna “Bruno Ubertini”Biobank of Veterinary Resource) under the Budapest Treaty underaccession number DPS RE RSCIC 16. The date of the deposit or transfer is28 Mar. 2019. The deposited microorganism was tested viable.

The term “IBV” refers to the infectious bronchitis virus which is wellknown to the person skilled in the art. The term “IBV” encompasses allstrains, genotypes, protectotypes, and serotypes of infectiousbronchitis virus.

The term “being protective against virulent M41 challenge” means thatsaid IBV provides protection against virulent M41 challenge or infectionfor subjects vaccinated with said IBV.

In one specific aspect of the IBV according to the present invention theIBV is attenuated.

The term “attenuated” refers to a pathogen having a reduced virulence incomparison to the wildtype isolate. In the present invention, anattenuated IBV is one in which the virulence has been reduced so that itdoes not cause clinical signs of an IBV infection but is capable ofinducing an immune response in the target animal, but may also mean thatthe clinical signs are reduced in incidence or severity in animalsinfected with the attenuated IBV in comparison with a “control group” ofanimals infected with non-attenuated IBV and not receiving theattenuated virus. In this context, the term “reduce/reduced” means areduction of at least 10%, preferably 25%, even more preferably 50%,still more preferably 60%, even more preferably 70%, still morepreferably 80%, still more preferably 90%, even more preferably 95% andmost preferably of 100% as compared to the control group infected withnon-attenuated IBV as defined above. Thus, an attenuated IBV strain isone that is suitable for incorporation into an immunogenic compositioncomprising a modified live IBV.

In another specific aspect of the IBV according to the present inventionthe IBV is attenuated in one-day-old chickens.

In another specific aspect of the IBV according to the present inventionthe IBV is inactivated.

Any conventional inactivation method can be used for purposes of thepresent invention. Thus, inactivation can be performed by chemicaland/or physical treatments which are known to the person skilled in theart. Preferred inactivation methods include the addition of cyclizedbinary ethylenimine (BEI) including the addition of a solution of2-bromoethyleneamine hydrobromide (BEA), which has been cyclized tobinary ethylenimine (BEI). Preferred further chemical inactivationagents comprise but are not limited to Triton X-100, Sodiumdeoxycholate, Cetyltrimethylammonium bromide, β-Propiolactone,Thimerosal, Phenol and Formaldehyde (Formalin). However, theinactivation may also comprise a neutralization step. Preferredneutralization agents include but are not limited to sodium thiosulfate,sodium bisulfite and the alike.

Preferred formalin inactivation conditions include formalinconcentration between from about 0,02% (v/v)-2,0% (v/v), more preferablyfrom about 0.1% (v/v)-1,0% (v/v), still more preferably from about 0,15%(v/v)-0,8% (v/v), even more preferably from about 0,16% (v/v)-0,6%(v/v), and most preferably about 0,2% (v/v)-0,4% (v/v). Incubation timedepends on the resistance of the IBV. In general, the inaction processis performed until no growth of the IBV can be detected in a suitablecultivation system.

Preferably, the inactivated IBV of the present invention is formalininactivated, preferably using the concentrations as describedhereinabove.

The inactivated IBV of the invention may be incorporated into liposomesusing known technology such as that described in Nature, 1974, 252,252-254 or Journal of Immunology, 1978, 120, 1109-13. In anotherembodiment of the invention, the inactivated IBV of the invention may beconjugated to suitable biological compounds such as polysaccharides,peptides, proteins, or the like, or a combination thereof.

In another specific aspect of the IBV according to the present inventionthe IBV is a non-recombinant.

The term “non-recombinant” as used herein relates to a RNA genome (orRNA sequence, cDNA sequence or protein) having no modifications (such asinsertions, deletions, inversions, relocations or a point mutations)introduced recombinantly by human intervention. The term“non-recombinant” as used with respect to a virus, means a virus notproduced by recombinant artificial manipulation of the viral genome. Theterm “non-recombinant virus” excludes recombinant genetically modifiedviruses. The term “genetically engineered” refers to an IBV which hasbeen mutated by using “reverse genetic” approaches where either a viralcDNA or a chemically synthesized RNA was involved.

The term “protein”, “amino acid” and “polypeptide” are usedinterchangeably. The term “protein” refers to a sequence of amino acidscomposed of the naturally occurring amino acids as well as derivativesthereof. The naturally occurring amino acids are well known in the artand are described in standard text books of biochemistry. Within theamino acid sequence the amino acids are connected by peptide bonds.Further, the two ends of the amino acid sequence are referred to as thecarboxyl terminus (C-terminus) and the amino terminus (N-terminus). Theterm “protein” encompasses essentially purified proteins or proteinpreparations comprising other proteins in addition. Further, the termalso relates to protein fragments. Moreover, it includes chemicallymodified proteins. Such modifications may be artificial modifications ornaturally occurring modifications such as phosphorylation,glycosylation, myristylation and the like.

In another specific aspect of the IBV according to the present inventionthe IBV is of a Massachusetts genotype or serotype.

IBV strains can be classified by serotype and genotype. Serotypeclassification involves treatment of the virus with neutralizingantibodies, whereas genotype classification generally involves examiningthe sequence of the 51 (spike) protein. However, the different IBVstrains are well known to the person skilled in the art. Infectiousbronchitis virus was first discovered in the United States in the 1930s.

The first IBV serotype identified was Massachusetts and remained theonly serotype until the discovery of a different IBV seroptpye in 1956.Today, IBV Mass (Massachusetts) viruses can be identified in manycountries of the world.

The IBV strain Beaudette is of Massachusetts type and was derivedfollowing at least 150 passages in chick embryos. The IBV strainBeaudette was originally isolated by Beaudette and Hudson (J. Am. Vet.Med. A. 90, 51-60, 1937) and passaged in chicken embryos. OtherMassachusetts type IBV strains besides Beaudette are H120, H52, and M41.The H120 strain was passaged 120 times in embryonated chickens eggs.

It is in the general knowledge of a person skilled in the art where toobtain any IBV strains. IBV strains can be be commercially purchased,obtained from scientific institutes or the genomes can be synthesized ascomplementary DNA as IBV strains have been sequenced and the sequenceshave been published and are, thus, available. Furthermore, IBV strainscan be isolated from samples obtained from infected chickens. Themethods to isolate IBV strains and to characterize the IBV strains arewell known to the person skilled in the art. Valter Leonardo de Quadros2011 (Dissertation, Das Infektiöse Bronchitis Virus (IBV):Molekularbiologische Untersuchungen zur Diagnostik and zum Vorkommensowie zur Pathogenität des Genotyps IBV QX in spezifisch pathogenfreien(SPF) Broilern, Freie Universität Berlin) and Farsang et al. 2002 (AvianPathology 31: 229-236) describe how to isolate and differentiatedifferent IBV strains.

In another specific aspect of the IBV according to the present inventionthe IBV is a M41, H52 or H120 strain.

In another specific aspect of the IBV according to the present inventionthe IBV is a M41 strain.

In another specific aspect of the IBV according to the present inventionthe IBV is a H52 or H120 strain.

It is in the general knowledge of a person skilled in the art where toobtain IBV M41, H52 or H120. Exemparily, IBV H52 strains can becommercially purchased such as exemplary Nobilis IB H52 (MSD AnimalHealth), AviPro IB H52 (Lohmann Animal Health GmbH & Co. KG), Bronchovac(Ceva) and the alike. IBV H120 strains can be commercially purchasedsuch as exemplary BIORAL H120 (Boehringer Ingelheim), HatchPak IB H120(Boehringer Ingelheim), AviPro IB H120 (Lohmann Animal Health GmbH & Co.KG), Poulvac IB H120 (Zoetis) and the alike. IBV M41 strains can becommercially purchased such as exemplary Volvac IB Fit (BoehringerIngelheim). Further, McDonald et al 1980 (Avain Pathology 9:245-259)disclose that IBV H52 can be obtained by Central Veterinary LaboratoryRotterdam, Kusters (J. gen Virol 68:343-352) disclose that IBV H52 canbe obtained by the Poultry Health Institute Dorn in the Netherlands (GDAnimal Health) and Chen et al 2007 (Avian Pathology 36(4):269-274)disclose that IBV H52 can be obtained by the China Institute ofVeterinary Drug Control. Furthermore, IBV M41, H52 or H120 are used asvaccine strains for decades and, therefore, can be found and isolatedfrom the field. The methods to isolate IBV M41, H52 or H120 strains andto characterize the IBV H52 strains are well known to the person skilledin the art. Exemplary, IBV H52 strains can be characterized as describedin Zwaagstra et al 1992 (J. Clin. Microbiol. 30 (1): 79-84), Handberg etal 1999 (Avian Pathology 28: 327-335) or Callison et al 2006 (Journal ofVirological Methods 138: 60-65). Zwaagstra et al 1992 and Handberg et al1999 for example disclose Massachusetts specifc Primers (for the S and Nprotein, respectively) for RT-PCR and sequencing and reference sequencesfor comparison. Further, H52 IBVs have been sequenced and the genomicsequences are available such as EU817497. Thus, the virus genome can begenerated by synthesizing its sequence and generated upon theapplication of reverse genetic systems.

Extended Cell or Tissue Tropism

In another specific aspect of the IBV according to the present inventionthe IBV has an extended cell or tissue tropism.

The term “cell or tissue” is known by the person skilled in the art. Theterm cell encompasses cell lines such as the cell lines listed elsewhereherein as well as primary cells. The term tissue encompasses cells fromtissues such as the ones listed elsewhere herein, exemplarily such asprimary chicken embryo cells from lung or liver or primary chickenfibroblasts. Encompassed is the propagation of cells or tissue (cells)in culture outside the organism. The term “culture” relates to thepropagation of cells (such as cell line cells or primary cells or tissuecells) outside the organism under defined culture conditions known bythe person skilled in the art.

The term “extended tropism” means that the IBV of the invention can bepropagated in cells (such as cell lines) or tissue cells (in addition toprimary chicken embryo cells from kidney). In contrast, IBV vaccinesdescribed in the prior art or non-cell adapted wildtype IBVs (celladapted IBV Beaudette strains are described) can only be propagated inembryonated chicken eggs or primary chicken embryo cells from kidney(after adaption). Accordingly, an IBV of the invention with extendedcell or tissue tropism has the capacity to infect and/or replicate inone or more cell lines or tissue cells other than primary chicken embryocells from kidney. Preferebly, the IBV of the invention with extendedcell or tissue tropism has the capacity to infect and/or replicate inone or more cell lines as listed herein. Accordingly, an IBV withextended cell or tissue tropism may, for example, have the capacity toinfect and/or replicate in PBS-12SF or HEK 293T cells.

Advantageously, the experimental data show that the deposited strain andits decendants have an extended cell or tissue tropism, they are capableto infect and replicate in different cell lines and tissue cells.

In another specific aspect of the IBV according to the present inventionthe IBV is infecting and/or replicating in at least one cell line orcell selected from the list consisting of: primary chicken embryo cellsfrom lung or liver or primary chicken fibroblasts, a chicken embryofibroblast cell line, a duck embryonic stem cell line, a human embryonickidney cell line, a baby hamster kidney cell line, an african greenmonkey kidney cell line, a rabbit kidney cell line, a canine kidney cellline, a chicken liver cell line, a bovine kidney cell line, a porcinekidney cell line and an insect cell line.

In another specific aspect of the IBV according to the present inventionthe IBV is infecting and/or replicating in at least one cell lineselected from the list consisting of: DF-1 (Douglas Foster), EB66 (duckembryonic stem cell line), PBS-12, PBS-12SF (PBS-12 serum free), BHK21(baby hamster kidney), HEK 293T (human embryonic kidney), Vero (VerdaReno), MA104, RK13 (rabbit kidney), LMH (leghorn male hepatoma), MDCK(Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15(porcine kidney), PK2A (porcine kidney), SF9, SF21 and SF+ (Spodopterafrugiperda).

In another specific aspect of the IBV according to the present inventionthe IBV is infecting and/or replicating in at least one cell lineselected from the list consisting of: DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104, MDCK, SF9 and RK13.

All mentioned cell lines are well known to the person skilled in the artand are commercially and/or publicly available. MDCK cells areexemplarily deposited at the American Tissue Culture Collection underaccession number ATCC CCL-34 or ATCC CRL-2285. DF-1 cells areexemplarily deposited at the American Tissue Culture Collection underaccession number ATCC CRL-12203. PBS-12SF cells are exemplarilydeposited at the American Tissue Culture Collection under accessionnumber ATCC PTA-8565 or deposited at RRID under CVCL_1K17. BHK-21 cellsare exemplarily deposited at the American Tissue Culture Collectionunder accession number ATCC CCL-10. HEK 293T cells are exemplarilydeposited at the American Tissue Culture Collection under accessionnumber ATCC CRL-3216. Vero cells are exemplarily deposited at theAmerican Tissue Culture Collection under accession number ATCC CCL-81.MA104 and cells are exemplarily deposited at the American Tissue CultureCollection under accession number ATCC CRL-2378. RK13 cells areexemplarily deposited at the American Tissue Culture Collection underaccession number ATCC CCL-37. SF9 cells are exemplarily deposited at theAmerican Tissue Culture Collection under accession number ATCC CRL-3357or ATCC PTA-3099.

Preferably, the IBV is infecting and/or replicating in the EB66, BHK,Vero, MA104, MDCK, SF9 and RK13 cell line.

In another specific aspect of the IBV according to the present inventionthe primary chicken embryo cell is a fibroblast or a cell derived fromliver or lung tissue.

Functional Definition—Protection

In another specific aspect of the IBV according to the present inventionthe IBV is protective against virulent M41 challenge or infection.

The term “protective” or “protective immunological response” or“protective immunity” is defined elsewhere herein.

In another specific aspect of the IBV according to the present inventionthe protection against virulent M41 challenge or infection is to bedetermined by ciliostasis score, reduced respiratory clinical signs,reduced viral RNA load in kidney tissue or reduced virus sheeding.

In another specific aspect of the IBV according to the present inventionthe attenuation of the IBV is increased compared to an IBV withoutextended cell or tissue tropism.

The term “increased attenuation” means, that the efficacy parameter (inparticular ciliostasis, but also rales, egg drop, kidney lesions, waterydiarrhea, weight loss, virus load or viral shedding) is reduced by atleast 10%, preferably by at least 20%, more preferably by at least 30%,even more preferably by at least 40%, even more preferably by at least50%, even more preferably by at least 60%, even more preferably by atleast 70%, even more preferably by at least 80%, even more preferably byat least 90%, even more preferably by at least 95% and most preferablyby 100% as compared to a subject of the same species immunized with anIBV without extended cell or tissue tropism. It is in the generalknowledge of a person skilled in the art how to measure the improvementin the efficacy parameters.

In another specific aspect of the IBV according to the present inventionthe attenuation of the IBV is increased compared to an IBV M41 withoutextended cell or tissue tropism.

In another specific aspect of the IBV according to the present inventionthe attenuation of the IBV is increased upon application in 1-day oldchickens compared to an IBV without extended cell or tissue tropism.

In another specific aspect of the IBV according to the present inventionthe attenuation of the IBV is increased upon application in 1-day oldchickens compared to an IBV M41 without extended cell or tissue tropism.

All Identifying Characteristics

In another specific aspect of the IBV according to the present inventionall identifying characteristics of the deposited IBV means that said IBVis attenuated, has an extended cell or tissue tropism and is protectiveagainst virulent M41 challenge or infection.

In another specific aspect of the IBV according to the present inventionall identifying characteristics of the deposited IBV means that the IBVis attenuated, has an extended cell or tissue tropism and the same orsimilar protection profile as the deposited IBV.

In another specific aspect of the IBV according to the present inventionall identifying characteristics of the deposited IBV means that the IBVis attenuated, is infecting and/or replicating in at least one cell lineselected from the list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13 and has the same or similar protectionprofile as the deposited IBV.

In another specific aspect of the IBV according to the present inventionall identifying characteristics of the deposited IBV means that the IBVis attenuated, is infecting and/or replicating in at least one cell lineselected from the list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13 and is protective against virulent M41challenge or infection.

Descendant

In another specific aspect of the IBV according to the present inventionthe descendant comprises up to 15 passages in cell culture of the IBVdeposited with the BVR of IZSLER under accession number DPS RE RSCIC 16.

In another specific aspect of the IBV according to the present inventionthe descendant comprises up to 10 passages in cell culture of the IBVdeposited with the BVR of IZSLER under accession number DPS RE RSCIC 16.

In another specific aspect of the IBV according to the present inventionthe descendant comprises up to 5 passages in cell culture of the IBVdeposited with the BVR of IZSLER under accession number DPS RE RSCIC 16.

Advantageously, the experimental data show that the deposited strain,but also its decendants have an extended cell or tissue tropism, theyare capable to infect and/or replicate in different cell lines andtissue cells. Surprisingly, the deposited strain and its decendantsmaintain vaccine efficacy until at least several passages and have animproved safety.

In another specific aspect of the IBV according to the present inventionsaid descendant IBV is attenuated, has an extended cell or tissuetropism and is protective against virulent M41 challenge or infection.

In another specific aspect of the IBV according to the present inventionsaid descendant IBV is attenuated, has an extended cell or tissuetropism and the same or similar protection profile as the deposited IBV.

In another specific aspect of the IBV according to the present inventionsaid descendant IBV is attenuated, is infecting and/or replicating in atleast one cell line selected from the list consisting of DF-1, EB66,PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13 and has the sameor similar protection profile as the deposited IBV.

In another specific aspect of the IBV according to the present inventionsaid descendant IBV is attenuated, is infecting and/or replicating in atleast one cell line selected from the list consisting of DF-1, EB66,PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13 and is protectiveagainst virulent M41 challenge or infection.

Nucleotide Sequences and Plasmids

Further, the present invention provides a nucleotide sequence encodingthe IBV as described herein. Thus, the present invention provides anucleotide sequence encoding an IBV (infectious bronchitis virus)deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16,any attenuated descendant IBV thereof having an extended cell or tissuetropism and being protective against virulent M41 challenge or anyattenuated IBV having an extended cell or tissue tropism and beingprotective against virulent M41 challenge.

Further, the present invention provides a plasmid comprising anucleotide sequence encoding the IBV as described herein. Thus, thepresent invention provides a plasmid comprising a nucleotide sequenceencoding an IBV (infectious bronchitis virus) deposited with the BVR ofIZSLER under accession number DPS RE RSCIC 16, any attenuated descendantIBV thereof having an extended cell or tissue tropism and beingprotective against virulent M41 challenge or any attenuated IBV havingan extended cell or tissue tropism and being protective against virulentM41 challenge.

The term “nucleic acid” or “nucleic acid sequence” or “nucleotidesequence” refers to polynucleotides including DNA molecules, RNAmolecules, cDNA molecules or derivatives. The term encompasses single aswell as double stranded polynucleotides. The nucleic acid of the presentinvention encompasses isolated polynucleotides (i.e. isolated from itsnatural context) and genetically modified forms. Moreover, comprised arealso chemically modified polynucleotides including naturally occurringmodified polynucleotides such as glycosylated or methylatedpolynucleotides or artificially modified ones such as biotinylatedpolynucleotides. Further, the terms “nucleic acid” and “polynucleotide”are interchangeable and refer to any nucleic acid. The terms “nucleicacid” and “polynucleotide” also specifically include nucleic acidscomposed of nucleotides with the nucleobases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine anduracil).

The term “plasmid” refers to cytoplasmic DNA that replicatesindependently of the bacterial chromosome within a bacterial host cell.

Cell

Further, the present invention provides a cell comprising the IBV orplasmid as described herein. The cell can be an eukaryotic orprokaryotic cell.

In another specific aspect of the cell according to the presentinvention the cell is a cell line or cell selected from the listconsisting of: primary chicken embryo cells, a chicken embryo fibroblastcell line, a duck embryonic stem cell line, a human embryonic kidneycell line, a baby hamster kidney cell line, an African green monkeykidney cell line, a rabbit kidney cell line, a canine kidney cell line,a chicken liver cell line, a bovine kidney cell line, a porcine kidneycell line and an insect cell line.

In another specific aspect of the cell according to the presentinvention the cell is a cell line selected from the list consisting of:DF-1 (Douglas Foster), EB66 (duck embryonic stem cell line), PBS-12,PBS-12SF (PBS-12 serum free), BHK21 (baby hamster kidney), HEK 293T(human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbitkidney), LMH (leghorn male hepatoma), MDCK (Madin-Darby canine kidney),MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney), PK2A, SF9, SF21and SF+(Spodoptera frugiperda).

In another specific aspect of the cell according to the presentinvention the cell is a cell line selected from the list consisting of:DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

In another specific aspect of the cell according to the presentinvention the primary chicken embryo cell is a fibroblast or a cellderived from liver or lung tissue.

Viral Particle, Immunogenic Composition and Vaccine

Further, the present invention provides a viral particle comprising theIBV as described herein.

Further, the present invention provides an immunogenic compositioncomprising the IBV as described herein.

Further, the present invention provides a vaccine comprising the IBV asdescribed herein.

Further, the present invention provides a modified live vaccine with anextended cell or tissue tropism comprising the IBV as described herein.

The term “immunogenic composition” refers to a composition thatcomprises at least one antigen, which elicits an immunological responsein the host to which the immunogenic composition is administered. Suchimmunological response may be a cellular and/or antibody-mediated immuneresponse to the immunogenic composition of the invention. Preferably,the immunogenic composition induces an immune response and, morepreferably, confers protective immunity against one or more of theclinical signs of a IBV infection. The host is also described as“subject”. Preferably, any of the hosts or subjects described ormentioned herein is an avian or poultry.

Usually, an “immunological response” includes but is not limited to oneor more of the following effects: the production or activation ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells and/or gamma-delta T cells, directed specifically toan antigen or antigens included in the immunogenic composition of theinvention. Preferably, the host will display either a protectiveimmunological response or a therapeutically response.

A “protective immunological response” or “protective immunity” will bedemonstrated by either a reduction or lack of clinical signs normallydisplayed by an infected host, a quicker recovery time and/or a loweredduration of infectivity or lowered pathogen titer in the tissues or bodyfluids or excretions of the infected host.

In case where the host displays a protective immunological response suchthat resistance to new infection will be enhanced and/or the clinicalseverity of the disease reduced, the immunogenic composition isdescribed as a “vaccine”.

The term “modified live” and “attenuated” are used interchangeableherein.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine comprises a pharmaceutically acceptable carrier.

The term “pharmaceutical-acceptable carrier” includes any and allsolvents, dispersion media, coatings, stabilizing agents, diluents,preservatives, antibacterial and antifungal agents, isotonic agents,adsorption delaying agents, adjuvants, immune stimulants, andcombinations thereof.

“Diluents” can include water, saline, dextrose, ethanol, glycerol, andthe like. Isotonic agents can include sodium chloride, dextrose,mannitol, sorbitol, and lactose, among others. Stabilizers includealbumin and alkali salts of ethylendiamintetracetic acid, among others.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the pharmaceutically acceptablecarrier is phosphate buffered saline.

Preferably, the immunogenic composition further comprises sucrosegelatin stabilizer.

Preferably, the pharmaceutically acceptable carrier is chitosan.

Chitosan is a natural deacetylated polysaccharide from chitin incrustaceans (e.g., shrimp, crab), insects, and other invertebrates.Recently, Rauw et al. 2009 (Vet Immunol Immunop 134:249-258)demonstrated that chitosan enhanced the cellular immune response of liveNewcastle disease vaccine and promoted its protective effect. Further,Wang et al., 2012 (Arch Virol (2012) 157:1451-1461) have shown resultsrevealing the potential of chitosan as an adjuvant for use in a liveattenuated influenza vaccine.

Preferably, the immunogenic composition can further include one or moreother immunomodulatory agents such as, e.g. interleukins, interferons,or other cytokines. The amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan.

In some aspects, the immunogenic composition of the present inventioncontains an adjuvant. “Adjuvants” as used herein, can include aluminumhydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21(Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (GalenicaPharmaceuticals, Inc., Birmingham, Ala.), water-in-oil emulsion,oil-in-water emulsion, water-in-oil-in-water emulsion. The emulsion canbe based in particular on light liquid paraffin oil (EuropeanPharmacopea type); isoprenoid oil such as squalane or squalene; oilresulting from the oligomerization of alkenes, in particular ofisobutene or decene; esters of acids or of alcohols containing a linearalkyl group, more particularly plant oils, ethyl oleate, propyleneglycol di-(caprylate/caprate), glyceryl tri-(caprylate/caprate) orpropylene glycol dioleate; esters of branched fatty acids or alcohols,in particular isostearic acid esters. The oil is used in combinationwith emulsifiers to form the emulsion. The emulsifiers are preferablynonionic surfactants, in particular esters of sorbitan, of mannide (e.g.anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycoland of oleic, isostearic, ricinoleic or hydroxystearic acid, which areoptionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymerblocks, in particular the Pluronic products, especially L121. See Hunteret al., The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.), JohnWiley and Sons, NY, pp 51-94 (1995) andTodd et al., Vaccine 15:564-570 (1997). Exemplary adjuvants are the SPTemulsion described on page 147 of “Vaccine Design, The Subunit andAdjuvant Approach” edited by M. Powell and M. Newman, Plenum Press,1995, and the emulsion MF59 described on page 183 of this same book.

A further instance of an adjuvant is a compound chosen from the polymersof acrylic or methacrylic acid and the copolymers of maleic anhydrideand alkenyl derivative. Advantageous adjuvant compounds are the polymersof acrylic or methacrylic acid which are cross-linked, especially withpolyalkenyl ethers of sugars or polyalcohols. These compounds are knownby the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Personsskilled in the art can also refer to U.S. Pat. No. 2,909,462 whichdescribes such acrylic polymers cross-linked with a polyhydroxylatedcompound having at least 3 hydroxyl groups, preferably not more than 8,the hydrogen atoms of at least three hydroxyls being replaced byunsaturated aliphatic radicals having at least 2 carbon atoms. Thepreferred radicals are those containing from 2 to 4 carbon atoms, e.g.vinyls, allyls and other ethylenically unsaturated groups. Theunsaturated radicals may themselves contain other substituents, such asmethyl. The products sold under the name Carbopol; (BF Goodrich, Ohio,USA) are particularly appropriate. They are cross-linked with an allylsucrose or with allyl pentaerythritol. Among then, there may bementioned Carbopol 974P, 934P and 971P. Most preferred is the use ofCarbopol 971P. Among the copolymers of maleic anhydride and alkenylderivative, are the copolymers EMA (Monsanto), which are copolymers ofmaleic anhydride and ethylene. The dissolution of these polymers inwater leads to an acid solution that will be neutralized, preferably tophysiological pH, in order to give the adjuvant solution into which theimmunogenic, immunological or vaccine composition itself will beincorporated.

Further suitable adjuvants include, but are not limited to, the RIBIadjuvant system (Ribi Inc.), Block co-polymer (CytRx, Atlanta Ga.),SAF-M (Chiron, Emeryville Calif.), monophosphoryl lipid A, Avridinelipid-amine adjuvant, heat-labile enterotoxin from E. coli (recombinantor otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, ornaturally occurring or recombinant cytokines or analogs thereof orstimulants of endogenous cytokine release, among many others.

It is expected that an adjuvant can be added in an amount of about 100μg to about 10 mg per dose, preferably in an amount of about 100 μg toabout 10 mg per dose, more preferably in an amount of about 500 μg toabout 5 mg per dose, even more preferably in an amount of about 750 μgto about 2.5 mg per dose, and most preferably in an amount of about 1 mgper dose. Alternatively, the adjuvant may be at a concentration of about0.01 to 50%, preferably at a concentration of about 2% to 30%, morepreferably at a concentration of about 5% to 25%, still more preferablyat a concentration of about 7% to 22%, and most preferably at aconcentration of 10% to 20% by volume of the final product.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine is effective in the treatment and/or prophylaxis of clinicalsigns caused by IBV in a subject of need. The terms “treatment and/orprophylaxis”, “clinical signs” and “of need” have been definedelsewhere.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention said immunogenic composition orvaccine is formulated for a single-dose administration.

The volume for a single-dose has been defined elsewhere herein.

It has furthermore been shown that one dose of the immunogeniccomposition of the present invention is effective after theadministration of such single dose of such immunogenic composition orvaccine.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine is administered subcutaneously, intramuscularly, oral, in ovo,via spray, via drinking water or by eye drop.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine comprises 1 to 10 log₁₀ EID₅₀ per dose of the IBV.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine comprises 2 to 5 log₁₀ EID₅₀ per dose of the IBV.

In another specific aspect of the immunogenic composition or vaccineaccording to the present invention the immunogenic composition orvaccine comprises 2 to 4 log₁₀ EID₅₀ per dose of the IBV.

Method for Culture

Further, the present invention provides a method for culturing an IBV ina cell line or tissue cell comprising the use of the IBV as describedherein. Thus, the present invention provides a method for culturing anIBV in a cell line or tissue cell comprising the use of the IBVdeposited with the BVR of IZSLER under accession number DPS RE RSCIC 16,any attenuated descendant IBV thereof having an extended cell or tissuetropism and being protective against virulent M41 challenge or anyattenuated IBV having an extended cell or tissue tropism and beingprotective against virulent M41 challenge.

In another specific aspect of the method for culturing an IBV in a cellline or tissue cell according to the present invention the IBV isinfecting and/or replicating in a cell line or tissue cell as describedherein.

In another specific aspect of the method for culturing an IBV in a cellline or tissue cell according to the present invention the cell line ortissue cell is selected from the list consisting of: primary chickenembryo cells, a chicken embryo fibroblast cell line, a duck embryonicstem cell line, a human embryonic kidney cell line, a baby hamsterkidney cell line, an African green monkey kidney cell line, a rabbitkidney cell line, a canine kidney cell line, a chicken liver cell line,a bovine kidney cell line, a porcine kidney cell line and an insect cellline.

In another specific aspect of the method for culturing an IBV in a cellline or tissue cell according to the present invention the cell line isselected from the list consisiting of: DF-1 (Douglas Foster), EB66 (duckembryonic stem cell line), PBS-12, PBS-12SF (PBS-12 serum free), BHK21(baby hamster kidney), HEK 293T (human embryonic kidney), Vero (VerdaReno), MA104, RK13 (rabbit kidney), LMH (leghorn male hepatoma), MDCK(Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15(porcine kidney), PK2A, SF9, SF21 and SF+(Spodoptera frugiperda).

In another specific aspect of the method for culturing an IBV in a cellline or tissue cell according to the present invention the cell line isselected from the list consisting of: DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13.

Kits

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration preferably foradministration to subjects, especially poultry. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for administration.

The present invention provides a kit comprising the IBV or theimmunogenic composition or vaccine as described herein.

In one specific aspect of the kit according to the present invention thekit further comprises an instruction letter for the treatment and/orprophylaxis of diseases of avians.

In one specific aspect of the kit according to the present invention thekit further comprises an instruction letter for the treatment and/orprophylaxis of diseases of poultry.

In one specific aspect of the kit according to the present invention thekit further comprises an instruction letter for the treatment and/orprophylaxis of IB.

In one specific aspect of the kit according to the present invention thekit further comprises a dispenser capable of administering a vaccine tosaid animal.

Method of Treatment

Further, the present invention provides a method for immunizing asubject comprising administering to such subject an immunogeniccomposition as described herein.

The term “immunizing” relates to an active immunization by theadministration of an immunogenic composition to a subject to beimmunized, thereby causing an immunological response against the antigenincluded in such immunogenic composition.

Preferably, immunization results in lessening of the incidence of theparticular IBV infection in a flock or in the reduction in the severityof clinical signs caused by or associated with the particular IBVinfection.

Further, the immunization of a subject in need with the immunogeniccompositions as provided herewith, results in preventing infection of asubject by IBV infection. Even more preferably, immunization results inan effective, long-lasting, immunological-response against IBVinfection. It will be understood that the said period of time will lastmore than 1 month, preferably more than 2 months, preferably more than 3months, more preferably more than 4 months, more preferably more than 5months, more preferably more than 6 months. It is to be understood thatimmunization may not be effective in all subjects immunized. However,the term requires that a significant portion of subjects of a flock areeffectively immunized.

Preferably, a flock of subjects is envisaged in this context whichnormally, i.e. without immunization, would develop clinical signsnormally caused by or associated with an IBV infection. Whether thesubjects of a flock are effectively immunized can be determined withoutfurther ado by the person skilled in the art. Preferably, theimmunization shall be effective if clinical signs in at least 33%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, stillmore preferably in at least 95% and most preferably in 100% of thesubjects of a given flock are lessened in incidence or severity by atleast 10%, more preferably by at least 20%, still more preferably by atleast 30%, even more preferably by at least 40%, still more preferablyby at least 50%, even more preferably by at least 60%, still morepreferably by at least 70%, even more preferably by at least 80%, stillmore preferably by at least 90%, still more preferably by at least 95%and most preferably by 100% in comparison to subjects that are eithernot immunized or immunized with an immunogenic composition that wasavailable prior to the present invention but subsequently infected bythe particular IBV.

Further, the present invention provides a method of treating orpreventing clinical signs caused by IBV in a subject of need, the methodcomprises administering to the subject a therapeutically effectiveamount of an immunogenic composition or vaccine as described herein.

As shown in the Examples, the immunogenic composition or vaccine asprovided herein has been proven to be efficacious in treating orpreventing clinical signs caused by IBV in a subject.

The term “treating or preventing” refers to the lessening of theincidence of the particular IBV infection in a flock or the reduction inthe severity of clinical signs caused by or associated with theparticular IBV infection. Thus, the term “treating or preventing” alsorefers to the reduction of the number of subjects in a flock that becomeinfected with the particular IBV (=lessening of the incidence of theparticular IBV infection) or to the reduction of the severity ofclinical signs normally associated with or caused by a IBV infection orthe reduction of virus shedding after infection with the particular IBVor preventing or lessening egg drop in laying hens after infection withthe particular IBV in a group of subjects which subjects have receivedan effective amount of the immunogenic composition as provided herein incomparison to a group of subjects which subjects have not received suchimmunogenic composition.

The “treating or preventing” generally involves the administration of aneffective amount of the immunogenic composition of the present inventionto a subject or flock of subjects in need of or that could benefit fromsuch a treatment/prophylaxis. The term “treatment” refers to theadministration of the effective amount of the immunogenic compositiononce the subject or at least some subjects of the flock is/are alreadyinfected with such IBV and wherein such subjects already show someclinical signs caused by or associated with such IBV infection. The term“prophylaxis” refers to the administration of a subject prior to anyinfection of such subject with IBV or at least where such subject ornone of the subjects in a group of subjects do not show any clinicalsigns caused by or associated with the infection by such IBV. The terms“prophylaxis” and “preventing” are used interchangeable in thisapplication.

The term “an effective amount” as used herein means, but is not limitedto an amount of antigen, that elicits or is able to elicit an immuneresponse in a subject. Such effective amount is able to lessen theincidence of the particular IBV infection in a flock or to reduce theseverity of clinical signs of the particular IBV infection.

Preferably, clinical signs are lessened in incidence or severity by atleast 10%, more preferably by at least 20%, still more preferably by atleast 30%, even more preferably by at least 40%, still more preferablyby at least 50%, even more preferably by at least 60%, still morepreferably by at least 70%, even more preferably by at least 80%, stillmore preferably by at least 90%, still more preferably by at least 95%and most preferably by 100% in comparison to subjects that are eithernot treated or treated with an immunogenic composition that wasavailable prior to the present invention but subsequently infected bythe particular IBV.

The term “clinical signs” as used herein refers to signs of infection ofa subject from IBV. Examples for such clinical signs include but are notlimited to respiratory distress, nephritis, salphingitis, abnormal eggproduction, ruffled feathers, depression, reduced growth rates andreduced appetite. Signs of respiratory distress encompass respiratorysigns including gasping, coughing, sneezing, tracheal rales, nasal andocular discharge, tracheal lesions and ciliostasis in the trachea. Signsof nephritis encompass kidney lesions and watery diarrhea. Signs ofabnormal egg production encompass egg drop, eggs of smaller size,inferior shell, reduced internal egg quality, eggs with thin albumen andciliostasis in the oviduct. However, the clinical signs also include butare not limited to clinical signs that are directly observable from alive animal. Examples for clinical signs that are directly observablefrom a live animal include nasal and ocular discharge, coughing,gasping, sneezing, tracheal rales, ruffled feathers, conjunctivitis,weight loss, reduced growth rates, reduced appetite, dehydration, waterydiarrhea, lameness, lethargy, wasting and unthriftiness and the like.

Preferably, the clinical signs lessened in incidence or severity in atreated subject compared to subjects that are either not treated ortreated with an immunogenic composition that was available prior to thepresent invention but subsequently infected by the particular IBV referto a reduction of ciliostasis, a reduction of rales, a reduction of eggdrop, a reduction of kidney lesions, a reduction of watery diarrhea, areduction in weight loss, a lower virus load, a reduced viral shedding,or combinations thereof.

The term “in need” or “of need”, as used herein means that theadministration/treatment is associated with the boosting or improvementin health or clinical signs or any other positive medicinal effect onhealth of the subjects which receive the immunogenic composition inaccordance with the present invention.

Further, the present invention provides a method of reducing theciliostasis in a subject of need, in comparison to a subject of anon-immunized control group of the same species, the method comprisesadministering to the subject a therapeutically effective amount of animmunogenic composition or vaccine as described herein.

As shown in the Examples, the immunogenic composition or vaccine asprovided herein has been proven to be efficacious in reducingciliostasis.

The term “ciliostasis” is well known to the person skilled in that art.The surface of the trachea is covered with specialised epithelial cells,which are lined with numerous, motile, hair-like structures calledcilia. The term “ciliostasis” encompasses the reduction or loss of ciliaand/or loss or partial loss of ciliary activity (movement). Ciliostasismay be determined by examining the inner lining of the tracheal ringsfor the movement of the cilia. It is in the general knowledge of aperson skilled in the art how to determine the movemnet of the cilia inthe trachea.

Preferably, the movement of the cilia is not reduced from day 10 afterchallenge or infection, more preferably from day 5 after challenge orinfection, more preferably from day 4 after challenge or infection, morepreferably from day 3 after challenge or infection and most preferablyfrom day 1 or 2 after challenge or infection with the IBV as compared toa subject of a non-immunized control group of the same species.

The term “reduction of ciliostasis” means, that the ciliostasis isreduced by at least 10%, preferably by at least 20%, more preferably byat least 30%, even more preferably by at least 40%, even more preferablyby at least 50%, even more preferably by at least 60%, even morepreferably by at least 70%, even more preferably by at least 80%, evenmore preferably by at least 90%, even more preferably by at least 95%and most preferably by 100% as compared to a subject of a non-immunizedcontrol group of the same species. It is in the general knowledge of aperson skilled in the art how to measure the reduction of theciliostasis.

Further, the present invention provides a method of reducing the viralRNA load in a subject of need, in comparison to a subject of anon-immunized control group of the same species, the method comprisingadministering to the subject a therapeutically effective amount of animmunogenic composition or vaccine as described herein.

The term “virus load” or “virus titer” is a measure of the severity ofan active viral infection, and can be determined by methods known to theperson skilled in the art. The term “viral titre” is a measure ofinfectious units per volume of a virus preparation. Viral titre is anendpoint in a biological procedure and is defined as the dilution atwhich a certain proportion of tests carried out in parallel show aneffect (Reed and Muench, 1938). The determination can be based on thedetection of viral proteins such as by antibody binding to the viralproteins and further detection or, alternatively, by detection of viralRNA by amplification methods such as RT-PCR. Monitoring of virionassociated viral RNA in plasma by nucleic acid amplification methods isa widely used parameter to assess the status and progression ofretroviral disease, and to evaluate the effectiveness of prophylacticand therapeutic interventions. Exemplary, the virus load or virus titercan be calculated by estimating the live amount of virus in an involvedbody fluid such as a number of RNA copies per milliliter of bloodplasma.

The term “reduction of viral RNA load” means, that the viral RNA load isreduced by at least 10%, preferably by at least 20%, more preferably byat least 30%, even more preferably by at least 40%, even more preferablyby at least 50%, even more preferably by at least 60%, even morepreferably by at least 70%, even more preferably by at least 80%, evenmore preferably by at least 90%, even more preferably by at least 95%and most preferably by 100% as compared to a subject of a non-immunizedcontrol group of the same species. It is in the general knowledge of aperson skilled in the art how to measure the reduction of viral RNAload.

Further, the present invention provides the immunogenic composition orvaccine as described herein for use in a method for immunizing asubject, the method comprises administering to the subject atherapeutically effective amount of said immunogenic composition orvaccine.

Further, the present invention provides the immunogenic composition orvaccine as described herein for use in a method of treating orpreventing clinical signs caused by IBV in a subject of need, the methodcomprises administering to the subject a therapeutically effectiveamount of said immunogenic composition or vaccine.

Further, the present invention provides the immunogenic composition orvaccine as described herein for use in a method of reducing theciliostasis in a subject of need, in comparison to a subject of anon-immunized control group of the same species, the method comprisesadministering to the subject a therapeutically effective amount of saidimmunogenic composition or vaccine.

Further, the present invention provides the immunogenic composition orvaccine as described herein for use in a method of reducing the viralRNA load in a subject of need, in comparison to a subject of anon-immunized control group of the same species, the method comprisingadministering to the subject a therapeutically effective amount of animmunogenic composition or vaccine.

In one specific aspect of the method or use according to the presentinvention said subject is avian.

The term “avian” is well known to the person skilled in the art. Theterm “avian” encompasses all birds including poultry.

In one specific aspect of the method or use according to the presentinvention said subject is poultry.

The term “poultry” is well known to the person skilled in the art. Theterm “poultry” encompasses chickens, turkeys, quails, pheasants,guineafowl, geese, and ducks. Further, the term “chicken” includesbroiler, laying hens, and reproductive stocks for both also referred asbreeders.

In one specific aspect of the method or use according to the presentinvention said subject is selected from the list consisting of chicken,turkey, quail, or pheasant.

In one specific aspect of the method or use according to the presentinvention said subject is chicken.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine is administered once.

It is understood, that a single-dose is administered only once. As shownin the Examples the immunogenic composition as provided herein has beenproven to be efficacious after the administration of a single dose to asubject of need.

The dose volume per poultry depends on the route of vaccination and theage of the poultry.

Typically, eye drop vaccines are administered in a volume of 1 to 100 μlper dose at any age. Preferably, the single-dose for eye drop vaccineshas a total volume between about 5 μl and 70 μl and more preferablybetween about 20 μl and 50 μl with a single 20 μl, 25 μl, 30 μl, 35 μl,40 μl, 45 μl or 50 μl dose being preferred. Most preferred, thesingle-dose for eye drop vaccines has a total volume between betweenabout 30 μl and 50 μl with a single 30 μl, 35 μl, 40 μl, 45 μl or 50 μldose being preferred.

Spray vaccines may contain the dose in a volume of 25 to 1000 μl forday-old poultry. Preferably, the single-dose for spray vaccines has atotal volume between about 50 μl and 5000 μl, more preferably betweenabout 75 μl and 2000 μl, more preferably between about 100 μl and 1000μl, even more preferably between about 200 μl and 900 μl, even morepreferably between about 300 μl and 800 μl and even more preferablybetween about 400 μl and 700 μl with a single 400 μl, 425 μl, 450 μl,475 μl, 500 μl, 525 μl, 550 μl, 575 μl, 600 μl, 625 μl, 650 μl, 675 μlor 700 μl dose being preferred. Most preferred the single-dose has atotal volume of 400 μl, 450 μl 500 μl, 550 μl, 600 μl, 650 μl or 700 μl.

The vaccine for in ovo vaccination may contain the dose in a volume of50 to 100 μl, preferably 50 μl. Preferably, the single-dose for in ovovaccines has a total volume between about 10 μl and 250 μl, morepreferably between about 15 μl and 200 μl, even more preferably betweenabout 20 μl and 150 μl, even more preferably between about 30 μl and 100μl, even more preferably between about 30 μl and 75 μl and with a single30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl or 75 μldose being preferred. Most preferred the single-dose has a total volumeof 40 μl, 45 μl, 50 μl, 55 μl or 60 μl.

The vaccine for intramuscular or subcutaneous vaccination or one dose ofa drinking water vaccine may contain the dose in a volume of 30 μl to1000 μl. Preferably, the single-dose has a total volume between about 30μl and 1000 μl, more preferably between about 50 μl and 500 μl, morepreferably between about 75 μl and 250 μl and even more preferablybetween about 100 μl and 200 μl with a single 100 μl, 110 μl, 120 μl,125 μl, 130 μl, 135 μl, 140 μl, 145 μl, 150 μl, 160 μl, 170 μl, 175 μl,180 μl, 190 μl, 155 μl, or 200 μl dose being the most preferred.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine is administered at twoor more doses.

However, the immunogenic composition can be administered at two or moredoses, with a first dose being administered prior to the administrationof a second (booster) dose.

In a preferred aspect of the two-time administration regimen, both thefirst and second doses of the immunogenic composition are administeredin the same amount. Preferably, each dose is in the preferred amountsspecified above. In addition to the first and second dose regimen, analternate embodiment comprises further subsequent doses. For example, athird, fourth, or fifth dose could be administered in these aspects.Preferably, subsequent third, fourth, and fifth dose regimens areadministered in the same amount as the first dose, with the time framebetween the doses being consistent with the timing between the first andsecond doses mentioned above.

Preferably, the first administration of the vaccine is performed withinthe first three weeks of age, more preferably within the first week ofage and most preferred at one day-of-age by methods as described below.A second administration can be performed within the first 20 weeks ofage, preferably within 16-18 weeks of age, more preferably between 6-12weeks of age. Exemplary, the iniatial (first) vaccination is performedat 1-10 days of age and the second vaccination (booster) is performedwith a live or inactivated vaccine at 6-12 or 16-18 weeks of age. Morepreferably, the iniatial (first) vaccination is performed at oneday-of-age and the second vaccination (booster) is performed with a liveor inactivated vaccine at 6-12 or 16-18 weeks of age.

In case in ovo vaccination is used, preferably the first admistration isperformed when embryos are between 15 to 19 days old, preferably at day17, 18 or 19, most preferably at day 18 of age. A second administrationcan be performed within the first three weeks of age, preferably withinthe first 10 days of age.

In one specific aspect of the method or use according to the presentinvention said immunogenic composition or vaccine is administeredsubcutaneously, intramuscularly, oral, in ovo, via spray, via drinkingwater or by eye drop.

The immunogenic composition is, preferably, administered topically orsystemically. Suitable routes of administration conventionally used areoral or parenteral administration, such as intranasal, intravenous,intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous,as well as inhalation, in ovo, via spray, via drinking water or by eyedrop. However, depending on the nature and mode of action of a compound,the immunogenic composition may be administered by other routes as well.For example, such other routes include intracutaneously, intravenously,intravascularly, intraarterially, intraperitnoeally, intrathecally,intratracheally, intracutaneously, intracardially, intralobally,intralobarly, intramedullarly, intrapulmonarily, intrarectally, andintravaginally. However, most preferred the immunogenic composition isadministered subcutaneously, intramuscularly, oral, in ovo, via spray,via drinking water or by eye drop.

Live IBV vaccines are preferably administered individually by eye drop,intranasal, intramuscular or subcutaneous.

More preferably, mass application methods, including drinking water andaerosol spray vaccination, are used. Also preferred is the use ofvaccines as embryo vaccines (so-called in ovo vaccines) as describedfurther below.

For example, broilers may be vaccinated at one-day of age or at 1-3weeks of age, particularly for broilers with high levels of MDA. Layingstock or reproduction stock may be vaccinated initially at 1-10 days ofage and boosted with the vaccine at 7-12 or 16-18 weeks of age.

As outlined above, the present invention also provides an IBV vaccinethat can be safely administered via the in ovo route and at the sametime is able to induce a protective immune response. The in ovoadministration is well known to the person skilled in the art and theperson skilled in the art can perform in ovo administration withoutfurther ado. The in ovo administration of the vaccine involves theadministration of the vaccine to an avian embryo while contained in theegg (for a review on in ovo vaccination see: Ricks et al., Advances inVet. Med. 495-515, 1999). The vaccine may be administered to anysuitable compartment of the egg (e. g. allantois fluid, yolk sac,amnion, air cell or into the embryo) as described in the art (Sharma;Am. J. Vet. Res. 45 1619-1623, 1984). Preferably the vaccine isadministered below the shell (aircell) membrane and chorioallantoicmembrane.

Preferably, the vaccine is injected into embryonated eggs during latestages of the embryonation, generally during the final quarter of theincubation period, preferably 3-4 days prior to hatch. Preferably, theadmistration is performed when embryos are between 15 to 19 days old,preferably at day 17, 18 or 19, most preferably at day 18 of age.Subsequently, the vaccinated embryonated eggs are transferred to anincubator for hatch. The process of in ovo administration can beautomated using a robotic injection process as described in the priorart.

Usually conventional vaccines for post-hatch vaccination of poultrycannot be used for in ovo vaccination, because late stage embryos arehighly susceptible to infection with most vaccine viruses examined.However, International patent application WO 01/64244 discloses that IBVvaccines can be used for in ovo administration provided it is applied ata very low doses. Further, Wakenell et al. 1986 (Am. J. Vet. Res., 47933-938) discloses that passaging an IB vaccine virus in tissue culturerendered the virus apathogenic for embryos.

In one specific aspect of the method or use according to the presentinvention said immunogenic composition or vaccine is administered viaeye drop.

Typically, the live vaccine for post-hatch administration comprises theattenuated IBV in a concentration of 10¹ to 10⁸ EID₅₀ (50% Egg InfectiveDose) per dose, preferably in a concentration of 10² to 10⁵ EID₅₀ perdose and, more preferably, in a concentration of 10² to 10⁴ EID₅₀ perunit dose and, even more preferably, in a concentration of 10² to 10³EID₅₀ per dose.

The live vaccine for in ovo administration typically comprises an amountof the attenuated IBV of 10² to 10⁷ EID₅₀/embryo, preferably 10² to 10³EID₅₀/embryo in a volume of 50 to 100 μl, preferably 50 μl.

Preferably, the immunogenic composition of the present inventioncomprises the IBV of the present invention in amounts of about 1 toabout 10 log₁₀ EID (egg infective dose)₅₀/ml per dose, preferably about2 to about 8 log₁₀ EID₅₀ per dose, preferably in an amount of about 2 toabout 7 log₁₀ EID₅₀ per dose, more preferably in an amount of about 2 toabout 6 log₁₀ EID₅₀ per dose, even more preferably in an amount of about2 to about 5 log₁₀ EID₅₀ per dose, even more preferably in an amount ofabout 2 to about 4 log₁₀ EID₅₀ per dose, most preferably in an amount ofabout 2 to about 3 log₁₀ EID₅₀ per dose. More preferably, theimmunogenic composition of the present invention comprises the IBV ofthe present invention in amounts of about 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 5.5, 6, 6.5, 7, 7.5 or log₁₀ EID₅₀ per dose.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine comprises 1 to 10 log₁₀EID₅₀ per dose of the IBV.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine comprises 2 to 5 log₁₀EID₅₀ per dose of the IBV.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine comprises 2 to 4 log₁₀EID₅₀ per dose of the IBV.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine is administered tosubjects within the first week of age, within the first three days ofage, within the first two days of age, or within the first day of age.

Preferably, the subject to be immunized is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days of age. Morepreferably, said subject to be immunized is 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 days of age. Most preferably, said subject to beimmunized is 1, 2, 3, 4, 5, 6 or 7 days of age.

However, it has to be understood that after vaccination of the subjectbeing a few days of age, it does need several days for the immune systemof the poultry to build up immunity against an IBV infection. Therefore,preferably, the subjects are immunized within the first 24 h of age.

In one specific aspect of the method or use according to the presentinvention the immunogenic composition or vaccine is administered tosubjects within the first day of age. As shown in the Examples theimmunogenic composition as provided herein has been proven to be safeand efficacious when administered to 1-day old poultry.

In one specific aspect of the method or use according to the presentinvention said method results in an improvement in an efficacy parameterselected from the group consisting of: prevention or reduction ofciliostasis, prevention or reduction of rales, prevention or reductionof egg drop, prevention or reduction of kidney lesions, prevention orreduction of watery diarrhea, prevention or reduction in weight loss, alower virus load, a reduced viral shedding or combinations thereof, incomparison to a subject of a non-treated control group of the samespecies.

The terms “treatment and/or prophylaxis” have been defined elsewhere,wherein the terms “prophylaxis” and “preventing” or “prevention” areused interchangeable in this application. Further, the terms “shedding”has been defined elsewhere, too.

The term “reducing”, “reduced”, “reduction” or “lower” means, that theefficacy parameter (ciliostasis, rales, egg drop, kidney lesions, waterydiarrhea, weight loss, virus load, viral shedding) is reduced by atleast 10%, preferably by at least 20%, more preferably by at least 30%,even more preferably by at least 40%, even more preferably by at least50%, even more preferably by at least 60%, even more preferably by atleast 70%, even more preferably by at least 80%, even more preferably byat least 90%, even more preferably by at least 95% and most preferablyby 100% as compared to a subject of a non-immunized control group of thesame species. It is in the general knowledge of a person skilled in theart how to measure the improvement in the efficacy parameters.

The term “virus load” has been defined elsewhere herein.

The term “ciliostasis” has been defined elsewhere herein.

The term “rales” is well known to the person skilled in that art.However, the term “rales” encompasses tracheal rales and refers tosounds emanating from the bronchi. Rales can be determined withoutfurther ado by the person skilled in the art.

The term “egg drop” is well known to the person skilled in that art. Theterm “egg drop” encompasses a decreased egg production.

In one specific aspect of the method or use according to the presentinvention the treatment or prevention results in a prevention orreduction of ciliostasis as compared to subjects of a non-treatedcontrol group of the same species.

In one specific aspect of the method or use according to the presentinvention the treatment or prevention results in a prevention orreduction of kidney lesions as compared to subjects of a non-treatedcontrol group of the same species.

In one specific aspect of the method or use according to the presentinvention the treatment or prevention results in a prevention orreduction of egg drop as compared to subjects of a non-treated controlgroup of the same species.

The present invention further provides the IBV, the viral particle orthe immunogenic composition or vaccine as described herein fortherapeutic use.

The present invention further provides the IBV or the viral particle asdescribed herein for use as an immunogen or vaccine.

The present invention further provides the IBV, the viral particle orthe immunogenic composition or vaccine as described herein for use as amedicament.

The present invention further provides the use of the IBV, the viralparticle or the immunogenic composition or vaccine as described hereinfor the manufacture of a medicament.

The present invention further provides the use of the IBV, the viralparticle or the immunogenic composition or vaccine as described hereinfor the treatment and/or prophylaxis of IBV infections in a subject.

In another specific aspect of the IBV according to the present inventionthe IBV is phenotypically stable. Advantageously, the experimental datashow that the deposited strain and its decendants are phenotypicallystable since the extended cell culture/tissue tropism and attenuationremains stable over time (over passage).

The term “phenotypically stable” means that the IBV remains itsfunctional features of having an extended cell culture/tissue tropismand being attenuated over time (over passage). Preferably, saidfunctional features are still present after at least 3 passages, morepreferably after at least 6 passages, even more preferably after atleast 9 passages, even more preferably after at least 12 passages, mostpreferred after 15 passages of the IBV in cell culture or tissueculture.

CLAUSES

The following clauses are also described herein:

1. An IBV (infectious bronchitis virus) deposited with the BVR of IZSLERunder accession number DPS RE RSCIC 16, any descendant IBV thereof orany IBV having all of the identifying characteristics of the IBVdeposited under DPS RE RSCIC 16.

2. An IBV (infectious bronchitis virus) deposited with the BVR of IZSLERunder accession number DPS RE RSCIC 16, any attenuated descendant IBVthereof having an extended cell or tissue tropism and being protectiveagainst virulent M41 challenge or any attenuated IBV having an extendedcell or tissue tropism and being protective against virulent M41challenge.

3. The IBV of clause 1 or 2, wherein the IBV is attenuated.

4. The IBV of any one of clauses 1 to 3, wherein the IBV is attenuatedin one-day-old chickens.

5. The IBV of any one of clauses 1 to 4, wherein the IBV isnon-recombinant.

6. The IBV of any one of clauses 1 or 5, wherein the IBV is of aMassachusetts genotype or serotype.

7. The IBV of any one of clauses 1 or 6, wherein the IBV is a M41, H52or H120 strain.

Extended Cell or Tissue Tropism

8. The IBV of any one of clauses 1 or 7, wherein the IBV has an extendedcell or tissue tropism.

9. The IBV of any one of clauses 1 to 8, wherein the IBV is infectingand/or replicating in a cell line or cell selected from the listconsisiting of: primary chicken embryo cells from lung or liver orprimary chicken fibroblasts, a chicken embryo fibroblast cell line, aduck embryonic stem cell line, a human embryonic kidney cell line, ababy hamster kidney cell line, an african green monkey kidney cell line,a rabbit kidney cell line, a canine kidney cell line, a chicken livercell line, a bovine kidney cell line, a porcine kidney cell line and aninsect cell line.

10. The IBV of any one of clauses 1 to 9, wherein the IBV is infectingand/or replicating in at least one cell line selected from the listconsisting of: DF-1 (Douglas Foster), EB66 (duck embryonic stem cellline), PBS-12, PBS-12SF (PBS-12 serum free), BHK21 (baby hamsterkidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104,RK13 (rabbit kidney), LMH (leghorn male hepatoma), MDCK (Madin-Darbycanine kidney), MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney),PK2A, SF9, SF21 and SF+(Spodoptera frugiperda).

11. The IBV of any one of clause 1 to 10, wherein the IBV is infectingand/or replicating in at least one cell line selected from the listconsisting of: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104,MDCK, SF9 and RK13.

12. The IBV of clause 9, wherein the primary chicken embryo cell is afibroblast or a cell derived from liver or lung tissue.

Functional Definition—Protection

13. The IBV of any one of clauses 1 to 12, wherein the IBV is protectiveagainst virulent M41 challenge or infection.

14. The IBV of clause 13, wherein the protection against virulent M41challenge or infection is to be determined by ciliostasis score, reducedrespiratory clinical signs, reduced viral RNA load in kidney tissue orreduced virus sheeding.

15. The IBV of any one of clauses 1 to 14, wherein the attenuation ofthe IBV is increased compared to an IBV without extended cell or tissuetropism.

16. The IBV of any one of clauses 1 to 15, wherein the attenuation ofthe IBV is increased compared to an IBV M41 without extended cell ortissue tropism.

17. The IBV of any one of clauses 1 to 16, wherein the attenuation ofthe IBV is increased upon application in 1-day old chickens compared toan IBV without extended cell or tissue tropism.

18. The IBV of any one of clauses 1 to 17, wherein the attenuation ofthe IBV is increased upon application in 1-day old chickens compared toan IBV M41 without extended cell or tissue tropism.

All Identifying Characteristics

19. The IBV of any one of clauses 1 to 18, wherein all identifyingcharacteristics of the deposited IBV means that said IBV is attenuated,has an extended cell or tissue tropism and is protective againstvirulent M41 challenge or infection.

20. The IBV of any one of clauses 1 to 19, wherein all identifyingcharacteristics of the deposited IBV means that the IBV is attenuated,has an extended cell or tissue tropism and the same or similarprotection profile as the deposited IBV.

21. The IBV of any one of clauses 1 to 20, wherein all identifyingcharacteristics of the deposited IBV means that the IBV is attenuated,is infecting and/or replicating in at least one cell line selected fromthe list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T,Vero, MA104 and RK13 and has the same or similar protection profile asthe deposited IBV.

22. The IBV of any one of clauses 1 to 21, wherein all identifyingcharacteristics of the deposited IBV means that the IBV is attenuated,is infecting and/or replicating in at least one cell line selected fromthe list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T,Vero, MA104 and RK13 and is protective against virulent M41 challenge orinfection.

Descendant

23. The IBV of any one of clauses 1 to 22, wherein said descendant IBVis attenuated, has an extended cell or tissue tropism and is protectiveagainst virulent M41 challenge or infection.

24. The IBV of any one of clauses 1 to 23, wherein said descendant IBVis attenuated, has an extended cell or tissue tropism and the same orsimilar protection profile as the deposited IBV.

25. The IBV of any one of clauses 1 to 24, wherein said descendant IBVis attenuated, is infecting and/or replicating in at least one cell lineselected from the list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13 and has the same or similar protectionprofile as the deposited IBV.

26. The IBV of any one of clauses 1 to 25, wherein said descendant IBVis attenuated, is infecting and/or replicating in at least one cell lineselected from the list consisting of DF-1, EB66, PBS-12, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13 and is protective against virulent M41challenge or infection.

27. A plasmid comprising a nucleotide sequence encoding the IBV of anyone of clauses 1 to 26.

28. A cell comprising the IBV or plasmid of any one of clauses 1 to 27.

29. The cell according to clause 28, wherein the cell is a cell line orcell selected from the list consisting of: primary chicken embryo cells,a chicken embryo fibroblast cell line, a duck embryonic stem cell line,a human embryonic kidney cell line, a baby hamster kidney cell line, anAfrican green monkey kidney cell line, a rabbit kidney cell line, acanine kidney cell line, a chicken liver cell line, a bovine kidney cellline, a porcine kidney cell line and an insect cell line.

30. The cell according to clauses 28 or 29, wherein the cell is a cellline selected from the list consisiting of: DF-1 (Douglas Foster), EB66(duck embryonic stem cell line), PBS-12, PBS-12SF (PBS-12 serum free),BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero(Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male hepatoma),MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15(porcine kidney), PK2A, SF9, SF21 and SF+(Spodoptera frugiperda).

31. The cell of any one of clauses 28 to 30, wherein the cell is a cellline selected from the list consisting of: DF-1, EB66, PBS-12SF, BHK,HEK 293T, Vero, MA104 and RK13.

32. The cell of clause 29, wherein the primary chicken embryo cell is afibroblast or a cell derived from liver or lung tissue.

33. A viral particle comprising the IBV of any one of clauses 1 to 26.

34. An immunogenic composition comprising the IBV of any one of clauses1 to 26.

35. A vaccine comprising the IBV of any one of clauses 1 to 26.

36. A modified live vaccine with an extended cell or tissue tropismcomprising the IBV of any one of clauses 1 to 26.

37. The immunogenic composition or vaccine of any one of clauses 34 to36, wherein the immunogenic composition or vaccine comprises apharmaceutically acceptable carrier.

38. The immunogenic composition or vaccine of clause 37, wherein thepharmaceutically acceptable carrier is phosphate buffered saline.

39. The immunogenic composition or vaccine of any one of clauses 34 to38, wherein the immunogenic composition or vaccine is effective in thetreatment and/or prophylaxis of clinical signs caused by IBV in asubject of need.

40. The immunogenic composition or vaccine of any one of clauses 34 to39, wherein the immunogenic composition or vaccine comprises 1 to 10log₁₀ EID₅₀ of the IBV per dose.

41. The immunogenic composition or vaccine of any one of clauses 34 to40, wherein the immunogenic composition or vaccine comprises 2 to 5log₁₀ EID₅₀ of the IBV per dose.

42. The immunogenic composition or vaccine of any one of clauses 34 to41, wherein the immunogenic composition or vaccine comprises 2 to 4log₁₀ EID₅₀ of the IBV per dose.

43. A kit comprising the IBV of any one of clauses 1 to 26 or theimmunogenic composition or vaccine of any one of clauses 34 to 42.

44. The kit according to clause 43, wherein the kit further comprises aninstruction letter for the treatment and/or prophylaxis of diseases ofavians.

45. The kit according to clause 43, wherein the kit further comprises aninstruction letter for the treatment and/or prophylaxis of diseases ofpoultry.

46. The kit according to clauses 43, wherein the kit further comprisesan instruction letter for the treatment and/or prophylaxis of IB.

47. A method for immunizing a subject comprising administering to suchsubject an immunogenic composition or vaccine according to any one ofclauses 34 to 42.

48. A method of treating or preventing clinical signs caused by IBV in asubject of need, the method comprising administering to the subject atherapeutically effective amount of an immunogenic composition orvaccine according to any one of clauses 34 to 42.

49. A method of reducing the ciliostasis in a subject of need, incomparison to a subject of a non-immunized control group of the samespecies, the method comprising administering to the subject atherapeutically effective amount of an immunogenic composition orvaccine according to any one of clauses 34 to 42.

50. A method of reducing the viral RNA load in a subject of need, incomparison to a subject of a non-immunized control group of the samespecies, the method comprising administering to the subject atherapeutically effective amount of an immunogenic composition orvaccine according to any one of clauses 34 to 42.

51. The immunogenic composition or vaccine according to any one ofclauses 34 to 42 for use in a method for immunizing a subject, themethod comprising administering to the subject a therapeuticallyeffective amount of said immunogenic composition or vaccine.

52. The immunogenic composition or vaccine according to any one ofclauses 34 to 42 for use in a method of treating or preventing clinicalsigns caused by IBV in a subject of need, the method comprisingadministering to the subject a therapeutically effective amount of saidimmunogenic composition or vaccine.

53. The immunogenic composition or vaccine according to any one ofclauses 34 to 42 for use in a method of reducing the ciliostasis in asubject of need, in comparison to a subject of a non-immunized controlgroup of the same species, the method comprising administering to thesubject a therapeutically effective amount of said immunogeniccomposition or vaccine.

54. The immunogenic composition or vaccine according to any one ofclauses 34 to 42 for use in a method of reducing the viral RNA load in asubject of need, in comparison to a subject of a non-immunized controlgroup of the same species, the method comprising administering to thesubject a therapeutically effective amount of said immunogeniccomposition or vaccine.

55. The method or use of any one of clauses 47 to 54, wherein saidsubject is avian.

56. The method or use of any one of clauses 47 to 55, wherein saidsubject is poultry.

57. The method or use of any one of clauses 47 to 56, wherein saidsubject is selected from the list consisting of chicken, turkey, quail,or pheasant.

58. The method or use of any one of clauses 47 to 57, wherein saidsubject is chicken.

59. The method or use of any one of clauses 47 to 58, wherein theimmunogenic composition or vaccine is administered once.

60. The method or use of any one of clauses 47 to 58, wherein theimmunogenic composition or vaccine is administered at two or more doses.

61. The method or use of any one of clauses 47 to 60, wherein saidimmunogenic composition or vaccine is administered subcutaneously,intramuscularly, oral, in ovo, via spray, via drinking water or by eyedrop.

62. The method or use of any one of clauses 47 to 61, wherein saidimmunogenic composition or vaccine is administered via eye drop.

63. The method or use of any one of clauses 47 to 62, wherein theimmunogenic composition or vaccine comprises 1 to 10 log₁₀ EID₅₀ perdose of the IBV.

64. The method or use of any one of clauses 47 to 63, wherein theimmunogenic composition or vaccine comprises 2 to 5 log₁₀ EID₅₀ per doseof the IBV.

65. The method or use of any one of clauses 47 to 64, wherein theimmunogenic composition or vaccine comprises 2 to 4 log₁₀ EID₅₀ per doseof the IBV.

66. The method or use of any one of clauses 47 to 65, wherein theimmunogenic composition or vaccine is administered to subjects withinthe first week of age, within the first three days of age, within thefirst two days of age, or within the first day of age.

67. The method or use of any one of clauses 47 to 66, wherein theimmunogenic composition or vaccine is administered to subjects withinthe first day of age.

68. The method or use of any one of clauses 47 to 67, wherein saidmethod results in an improvement in an efficacy parameter selected fromthe group consisting of: prevention or reduction of ciliostasis,prevention or reduction of rales, prevention or reduction of egg drop,prevention or reduction of kidney lesions, prevention or reduction ofwatery diarrhea, prevention or reduction in weight loss, a lower virusload, a reduced viral shedding or combinations thereof, in comparison toa subject of a non-treated control group of the same species.

69. The method or use of any one of clauses 47 to 68, wherein thetreatment or prevention results in a prevention or reduction ofciliostasis as compared to subjects of a non-treated control group ofthe same species.

70. The method or use of any one of clauses 47 to 69, wherein thetreatment or prevention results in a prevention or reduction of kidneylesions as compared to subjects of a non-treated control group of thesame species.

71. The method or use of any one of clauses 47 to 70, wherein thetreatment or prevention results in a prevention or reduction of egg dropas compared to subjects of a non-treated control group of the samespecies.

72. The IBV of any one of clauses 1 to 26, the viral particle of clause33 or the immunogenic composition or vaccine of any one of clauses 34 to42 for therapeutic use.

73. The IBV of any one of clauses 1 to 26 or the viral particle ofclause 33 for use as an immunogen or vaccine.

74. The IBV of any one of clauses 1 to 26, the viral particle of clause33 or the immunogenic composition or vaccine of any one of clauses 34 to42 for use as a medicament.

75. Use of the IBV of any one of clauses 1 to 26, the viral particle ofclause 33 or the immunogenic composition or vaccine of any one ofclauses 34 to 42 for the manufacture of a medicament.

76. Use of the IBV of any one of clauses 1 to 26, the viral particle ofclause 33 or the immunogenic composition or vaccine of any one ofclauses 34 to 42 for the treatment and/or prophylaxis of IBV infectionsin a subject.

77. The IBV of any one of clauses 1 to 26, wherein the descendantcomprises up to 15 passages in cell culture of the IBV deposited withthe BVR of IZSLER under accession number DPS RE RSCIC 16.

78. The IBV of any one of clauses 1 to 26, wherein the descendantcomprises up to 10 passages in cell culture of the IBV deposited withthe BVR of IZSLER under accession number DPS RE RSCIC 16.

79. The IBV of any one of clauses 1 to 26, wherein the descendantcomprises up to 5 passages in cell culture of the IBV deposited with theBVR of IZSLER under accession number DPS RE RSCIC 16.

80. The IBV of any one of clauses 1 to 26 or 77 to 79, wherein the IBVis phenotypically stable.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1.: Immunofluorescence staining for IB66HP-infected EB66® cells.

FIG. 2.: Immunofluorescence staining for IB66HP 72 hours after infectionof different cell lines. Negative controls were included but are notshown.

EXAMPLES

The following examples are set forth below to illustrate specificembodiments of the present invention. These examples are merelyillustrative and are understood not to limit the scope or the underlyingprinciples of the present invention.

Example 1 Adaption of IBV to Cells and In Vitro and In VivoCharacterization

An attenuated IBV M41 is used for 10 serial passages in EB66® cells. Thematerial harvested after each passage is characterized by EID₅₀ andTCID₅₀. Indeed, infective titers are detected already in the firstpassage after inoculation of EB66® cells with an attenuated IBV Massseed stock. Results for EID₅₀ (infectivity for embryonated eggs) andTCID₅₀ (infectivity for cell culture) titer determination for the first10 passages and are summarized in table 1 and detection ofimmunofluorescence signal for IB66HP infected EB66® cells in FIG. 1. Theresults confirm that the IBV is adapted to cell culture in EB66® cells.The IBV is efficiently adapted after one passage in EB66® cells, whereasthe infectivity for embryonated eggs is not affected. A passage 4 of theIBV (IB66HP) is deposited at the BVR of IZSLER (Biobanking of VeterinaryResources of the Istituto Zooprofilattico Sperimentale della Lombardia edell'Emilia Romagna “Bruno Ubertini”) under accession number DPS RERSCIC 16.

TABLE 1 Summary of EID₅₀ and TCID₅₀ titers throughout IB66HP passagingin EB66 ® cells. Passage 1 2 3 4 5 6 7 8 9 10 EID₅₀/ml 5.6 9 7.1 5.3 7 76.8 7.5 6.6 6.6 TCID₅₀/ml 1 6.5 4.25 5.5 6.3 6.32 4.6 7 6 5.4From table 1 and the other experiments further below, it is appearentthat IB66 HP is phenotypically stable since the extended cellculture/tissue tropism and attenuation remains over time (over passage).

Serial Passaging in EB66® Cells

EB66® cells are seeded at a density of 10⁶ cells/ml into a 250 ml shakerflask with a total volume of 50 ml in GRO I medium (SIGMA, cat.:141530C) supplemented with glutamine and 1 ml CHO feed (SIGMA, cat:1615). The cells are infected with a 1/10 dilution of attenuated IBVMass seed stock for the first passage which is incubated for 96 hours.For the following passages 2 to 10 inoculation with the harvestedculture is performed with a 1/10 dilution of the previous passage intofreshly seeded EB66® cells which are subsequently incubated for 48 to 96hours under the same cultivation conditions. A passage 4 (supernatant)was generated for deposit by inoculation of 4*10⁵ cells/ml with a 1/250dilution of passage 3 and was harvested 72 hours post infection and hetiter of the harvested material is determined by the 50% embryoinfectious dose (EID₅₀) and immunofluorescence tissue culture infectiousdose 50 (TCID₅₀) assay which are 10^(5.75)/ml and 10^(7.33)/ml,respectively.

Determination of the 50% Embryo Infectious Dose (EID₅₀)

For the determination of the EID₅₀ titer 10-day old embryonated SPFchicken eggs are inoculated with 100 μl 10-fold serial dilution per egginto the allantoic cavity in 6 replicates per dilution. Infected eggsare incubated for 7 days and candling is conducted every 24 h in orderto determine mortality. Eggs with dead embryos prior to 24 hours ofincubation are not included in the evaluation. The EID₅₀/mL iscalculated as described by Reed & Muench.

Determination of the 50% Tissue Culture Infectious Dose (TCID₅₀)

For the determination of the TCID₅₀, 2.5 to 3*10⁵ cells with a viabilityof at least 90% are seeded per well into a 96 well plate one day priorto infection with a 10-fold dilution series of passaged IBV Mass. Mediumis removed from the cells and they are infected with 100 μl per well in5 replicates per dilution. After 72 hours of static incubation at 37° C.and 7.5% CO₂, medium is removed and cells are fixed for 15 min at 4° C.with 50 μl 80% acetone per well. A washing step with 1×PBS is conductedand followed by incubation with 50 μl per well of a 1:1000 dilution ofanti-IBV Mass antiserum (Charles River Laboratories, Cat.: 10100454) at37° C. for 1 hour. The plates are washed twice with 1×PBS before 50 μlof a 1:2000 dilution of Alexa 488-conjugated anti-chicken antibody(Invitrogen, Cat.: A11039) are added and incubated at 37° C. for 1 hour.Subsequently, a final wash with 1×PBS is conducted and 100 μl of 1×PBSare added into each well followed by the evaluation in an invertedfluorescence microscope. The infectious titer of IBV in EB66® cells isdetermined according to the formula of Reed & Muench.

Infectivity of IB66HP for Different Cell Lines

Different cell lines are seeded into 96 well plates to reach 70-80%confluence the next day. Cells are infected with a 10-fold dilutionseries of an IB66HP P8 set to a titer of 10⁵ to 10^(5.75) TCID₅₀/mlderived from titration on EB66® cells. Medium is removed from the cellsand they are infected with 100 μl per well in 4 replicates per dilution.After 72 hours the TCID₅₀ titer is determined. Indeed, infectivity ofIB66HP for different primary cells and cell lines is detected andresults are listed in table 2 and 3 and shown in FIG. 2.

TABLE 2 IB66HP has an extended tissue tropism. TCID₅₀ determination invarious cell lines for IB66HP set to a titer of 10⁵ TCID₅₀ prior totitration. Cell Species Tissue Log TCID₅₀/ml EB66 duck Embryonic stem 5cell BHK hamster Kidney 4 MA104 African green Kidney 2 monkey Vero EUAfrican green Kidney 2.67 monkey RK13 rabbit Kidney 3 CEK Chicken embryoPrimary kidney 5.33 CEH Chicken embryo Primary liver 3.67 CEL Chickenembryo Primary lung 6

TABLE 3 IB66HP has an extended tissue tropism. TCID₅₀ determination invarious cell lines for IB66HP set to a titer of 10^(5.75) TCID₅₀ priorto titration. Cell Species Tissue Log TCID₅₀/ml SF9 Fall armyworm Pupalovarian tissue 5.6 CEF Chicken embryo Primary fibroblasts 4.5 MDCK Dogkidney 5.38

Conclusion Example 1

The attenuated IBV M41 was adapted to cell culture. The deposited strainas well as descendants show an extended cell or tissue tropism as theywere able to infect in primary cells and a broad range of different celllines.

Example 2 Determination of IB66HP Safety and Efficacy In Vivo

The attenuated IBV Mass allantoic fluid stock (P0) and passages 5 (P5)and 10 (P10) in EB66® cells are used to assess their safety and efficacyin 1-day-old SPF layer chickens against virulent M41 challenge.Throughout the whole study chickens are housed in isolation units andare provided with water and commercial balanced feed formula ad libitum.Chickens are observed daily for clinical signs. Three groups of chickensare vaccinated with P0, P5 and P10 each, at a dose of 10⁴ TCID₅₀/chickenvia eye drop. One group is treated with placebo and serves as challengecontrol group. Seven days post vaccination seven animals per groupvaccinated with P0, P5 or P10 are euthanized to assess the safety viascoring of the tracheal ciliostasis. For this, the trachea is removedand cut into transversal sections of which 3 of the lower, 4 of themiddle and 3 of the upper part are used for safety assessment. All ringsare evaluated by light microscopy for beating of the cilia. Each ring isscored individually as described in table 4.

TABLE 4 Ciliostasis scoring scheme for the assessment of safety ScoreCiliar activity [%] 0 100 1 75-99 2 50-74 3 25-49 4  0-24

Twenty-one days post vaccination all remaining chickens are challengedwith virulent IBV M41 with a dose of 10²⁵ EID₅₀/chicken via eye drop.Efficacy is assessed at seven days post vaccination by ciliostasisscoring as described in table 4. A ring is recorded as normal if morethan 50% of the internal ring shows vigorous ciliar movement (Score 2and lower). A ring is recorded as positive for ciliostasis if less than50% of the cilia are beating (Score 3 and 4). An animal is consideredprotected if not fewer than 9 out of 10 rings show normal ciliaractivity. Throughout the whole study, all animals are observed daily forclinical signs such as depression, respiratory, digestive orneurological signs, locomotive damage, prostration or ruffled feathers.

The aim of the study is to determine whether the cell culture adaptationand cell passage of IB66HP has an effect on safety and efficacy comparedto the parental IBV M41att allantoic fluid stock when applied to1-day-old chickens. The assessment of safety by clinical signs andciliostais scoring of tracheal explants revealed that P5 and P10 ofIB66HP in Eb66® cells have an improved safety profile compared to IBVM41att. The ciliostasis score are strongly reduced for IB66HP P5 and P10in contrast to IBV M41att (table 5). In addition, clinical signs in theanimals of groups vaccinated with IB66HP P5 and P10 were reducedcompared to the animals vaccinated with M41att. Conclusively, IB66HP ismore attenuated than M41att and has an improved safety profile forvaccination of 1-day-old chickens.

TABLE 5 Results for assessment of safety for IB66HP EB66 ® cell passage5 (P5) and 10 (P10) in comparison to the allantoic fluid stock of M41att5 days post vaccination. The mean ciliostasis score per group iscalculated by adding up the sum score of the individual chickens pergroup and dividing the group sum by the number of animals (highestpossible score 40, lowest possible score 0). For not affected animals,at least 9 of the 10 tracheal explants show normal ciliar activity(score ≤2). Mean ciliostasis #Animals/not Vaccination score affectedM41att P0 30.6 5/1 IB66HP P5 8.2 5/5 IB66HP P10 6.4 5/5 — 0.4 5/5

All animals of the non-vaccinated control group show clinical signs anda high ciliostasis score after challenge with virulent IBV M41. Incontrast, the EB66® cell passaged IB66HP P5 and P10 are able to reduceclinical signs and the damage of the trachea as efficiently as M41att(table 6).

TABLE 6 Results for assessment of efficacy for IB66HP EB66 ® cellpassage 5 (P5) and 10 (P10) in comparison to the allantoic fluid stockof M41att 7 days post challenge. The mean ciliostasis score per group iscalculated by adding up the sum score of the individual chickens pergroup and dividing the group sum by the number of animals (highestpossible score 40, lowest possible score 0). For not affected animals,at least 9 of the 10 tracheal explants show normal ciliar activity(score ≤2). % animals Mean ciliostasis #animals/Not with normalVaccination Challenge score affected ciliostasis M41att P0 M41 2.2 7/7100 IB66HP P5 M41 0.1 9/9 100 IB66HP P10 M41 3 10/9  90 — M41 35.9 9/0 0

In summary, IB66HP is an attenuated IBV strain that efficiently infectsand replicates in different cell lines and tissue cells. Surprisingly,it has an improved safety for application in 1-day-old chickens comparedto the parental egg-restricted IBV M41att strain. Furthermore, IB66HPmaintains vaccine efficacy. In contrast, the only other known cellculture adapted and highly attenuated IBV strain Beaudette does notconfer sufficient protection after challenge infection. Thus, IB66HP isthe first described IBV that is attenuated, has an extended cell cultureor tissue tropism and displays efficacy against virulent challenge afterapplication as a vaccine. Further, IB66HP has been shown to bephenotypically stable.

1. An IBV (infectious bronchitis virus) deposited with the BVR of IZSLERunder accession number DPS RE RSCIC 16, any descendant IBV thereof orany IBV having all of the identifying characteristics of the IBVdeposited under DPS RE RSCIC
 16. 2. An IBV (infectious bronchitis virus)deposited with the BVR of IZSLER under accession number DPS RE RSCIC 16,any attenuated descendant IBV thereof having an extended cell culture ortissue tropism and being protective against virulent M41 challenge orany attenuated IBV having an extended cell or tissue tropism and beingprotective against virulent M41 challenge.
 3. The IBV of claim 1,wherein the IBV is attenuated.
 4. The IBV of any one of claim 1, whereinthe IBV is non-recombinant.
 5. The IBV of any one of claim 1, whereinthe IBV is of a Massachusetts genotype or serotype.
 6. The IBV of anyone of claim 1, wherein the IBV has an extended cell or tissue tropism.7. The IBV of claim 1, wherein the IBV is infecting and/or replicatingin a cell line or cell selected from the list consisting of: primarychicken embryo cells from lung or liver or primary chicken fibroblasts,a chicken embryo fibroblast cell line, a duck embryonic stem cell line,a human embryonic kidney cell line, a baby hamster kidney cell line, anAfrican green monkey kidney cell line, a rabbit kidney cell line, acanine kidney cell line, a chicken liver cell line, a bovine kidney cellline, a porcine kidney cell line and an insect cell line.
 8. The IBV ofclaim 1, wherein the IBV is infecting and/or replicating in at least onecell line selected from the list consisting of: DF-1 (Douglas Foster),EB66 (duck embryonic stem cell line), PBS-12, PBS-12SF (PBS-12 serumfree), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney),Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn malehepatoma), MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovinekidney), PK15 (porcine kidney), PK2A, SF9, SF21 and SF+ (Spodopterafrugiperda).
 9. The IBV of claim 1, wherein the attenuation of the IBVis increased compared to an IBV without extended cell or tissue tropism.10. A plasmid comprising a nucleotide sequence encoding the IBV ofclaim
 1. 11. A cell comprising the IBV or plasmid of claim
 1. 12. Animmunogenic composition comprising the IBV of claim
 1. 13. Theimmunogenic composition of claim 12 wherein the immunogenic compositionis a vaccine.
 14. The immunogenic composition of claim 12 wherein theimmunogenic composition is a modified live vaccine.
 15. A method forimmunizing a subject comprising administering to such subject theimmunogenic composition of claim
 12. 16. A method of treating orpreventing clinical signs caused by IBV in a subject of need, the methodcomprising administering to the subject a therapeutically effectiveamount of the immunogenic composition of claim
 12. 17. A method ofreducing ciliostasis in a subject of need, in comparison to a subject ofa non-immunized control group of the same species, the method comprisingadministering to the subject a therapeutically effective amount of theimmunogenic composition of claim
 12. 18. The method of claim 15, whereinsaid subject is a chicken.
 19. The method of claim 15, wherein theimmunogenic composition is administered once.
 20. The method of claim15, wherein said immunogenic composition is administered subcutaneously,intramuscularly, oral, in ovo, via spray, via drinking water or by eyedrop.